Protein Engineering and Induce Mechanism of Raw-Starch- Digesting Amylase from Cytophaga sp.

• Main Authors: Rong-Jen Shiau, 蕭蓉禎
• Other Authors: Chii-Ling Jeang
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/41817791884681885530

博士 === 國立中興大學 === 食品科學系 === 92 === Starch, mostly found in plants, is the major energy source for living creatures. Industrially, starch granules from plants are subjected to gelatinization, a process which breaks starch granules into soluble starch, before hydrolyzed by amylases. However, the gelatinization is time and energy consuming. Raw starch digesting amylases which are capable to directly hydrolyze starch granules into small sugar molecules, therefore, become a great interest for this industrial purpose. Previously, a Cytophaga sp. which produces a raw starch digesting amylase (RSDA) was isolated in our laboratory. This RSDA demonstrates an excellent hydrolysis ability to starch granules. To further investigate the characters of RSDA, the recombinant RSDA expressed in E. coli was purified by the column chromatography and subjected to various biochemical analyses. The enzyme unit of 1 mL culture of E. coli and Cytophaga sp. was 240 and 246 unit, respectively. The purified recombinant RSDA displayed a specific activity of 3.1 × 104U/mg, which is 4.8-folds greater than the wild type RSDA. For hydrolysis of soluble starch, the optimal temperatures of recombinant and wild type RSDAs were 40 ℃ and 50 ℃, respectively. For hydrolysis of raw starch, the optimal pH values of recombinant and wild types RSDAs were pH 6.5 ~ 7.5 and pH 7.0, respectively. However, for hydrolysis of soluble starch, the optimal pH for both recombinant and wild type RSDAs were pH 4 ~ 6.5. Finally, the pH stability assays demonstrated that the recombinant RSDA is less stable than the wild type in both acid and alkaline solutions. In order to increase the applications of RSDA in food industry, a genetic engineering approach was used to improve the thermostability of RSDA. Using PCR based site-directed mutagenesis, a heat-stable mutant RSDA, in which both Arg178 and Gly179 residues were deleted, was generated. The activity assay showed that at 65 ℃ the half-life of this mutant RSDA significantly increased 20-fold, compared to the wild type RSDA. This enhancement is via a Ca2+ -independent mechanism. At pH 3.0, the mutant RSDA remained 41 % of enzymatic activity, however, the wild type RSDA lost activity completely. The isoelectro- focusing assays showed that the pI values of wild type and mutant RSDA were 8.5 and 8.3, respectively. The kinetic assays demonstrated that the mutation on RSDA resulted in decreasing its catalytic activity and the affinity to raw starch. Together, these data suggest that deletion of two residues from RSDA will decrease the positive charge of the protein molecule, increase the protein stability, and lead to enhance its thermostability. Since the expression of RSDA is only highly induced by raw starch but not by other sugars, an investigation about how raw starch affects the protein expressions in Cytophaga sp. was taken place. A proteomic approach was applied to identify the raw-starch regulated proteins. In a medium containing 0.2 % raw starch, the expression levels of 15 proteins were up-regulated, and 12 proteins were down-regulated, suggesting that raw starch not only induces, but also represses protein expression in Cytophaga sp.. Using MALDI-TOF Mass protein analysis, three up-regulated proteins were identified, which may be the 60 kDa chaperonin (Cpn60), glutaminase and pyruvate, phosphate dikinase (PPDK). The full-length (1,641 bp) of Cytophaga sp. cpn60 gene was cloned by using PCR technique. Amino acids comparison search revealed that Cpn60 showed the highest identity (84 %) to B. subtilis 58K GroEL. A recombinant expression vector contained a large portion of cpn60 (1,539 bp) was transformed into E. coli. This truncated rCpn60 was later produced in E. coli, purified, and subjected to the antibody preparation. The induction of Cpn60 in Cytophaga sp. by raw starch was confirmed by using Western blot analysis with anti-truncated rCpn60 antibody. The work present in this thesis revealed the biochemical characters of recombinant RSDA, the improvement of thermostability of RSDA, and most importantly, a new approach to study how Cytophaga sp. cells respond to raw starch stimulation.